It goes without saying that an optical microscope is an indispensable observation tool in the fields of natural science, engineering, and industry. Especially in recent years, a microscope having higher performance using laser as an illumination light source has been becoming essential in advanced technology development. As a representative example, a coherent anti-Stokes Raman scattering (CARS) microscope (Patent Literatures 1 and 2) is known. The CARS microscope irradiates a sample with two types of laser beam, pump light and Stokes light, for observation of anti-Stokes light caused as a result of resonance of difference frequency between these lights with natural frequency of the sample molecule (hereinbelow referred to as CARS light). The microscope, which enables quantitative analytic observation of materials in the sample with the spectrum of the CARS light, attracts attention as non-invasive quantitative analysis means.
The operation principle of the CARS microscope will be described. CARS is light emission by third-order nonlinear polarization. To cause CARS, the pump light, the Stokes light, and probe light are required. In many cases, to reduce the number of light sources, the probe light is substituted with the pump light. In this case, induced third-order polarization is represented as P(ω)=(χr(3)(ω)+χnr(3))EP2(ωP)E*S(ωS). Here χr(3)(ω) is a resonance section of the molecular vibrations of the third-order electric susceptibility, and χnr(3) is a non-resonance section. Further, the electric field of the pump light and the probe light is represented with EP, and the electric field of the Stokes light is represented with ES. The non-resonance section has no frequency dependence. The asterisk attached to ES indicates a complex conjugate. The intensity of the CARS light is the square of the absolute value of P(ω). A mechanism to cause the CARS light will be described using an energy level diagram of the molecule shown in FIG. 15. Numeral 1401 denotes a vibration ground state of the molecule and 1402 denotes a vibration excited state. The pump light with a frequency of ωP and the Stokes light with a frequency of ωS are irradiated simultaneously. At this time, the molecule is excited via a virtual level 1403 to a vibration excitation level in the state 1402. When the molecule in the excitation state is irradiated with the probe light with a frequency of ωP, the molecule returns to the vibration ground state while emitting the CARS light with a frequency of ωAS via the virtual level 1404. The frequency of the CARS light at this time is represented as ω=2ωP−ωS.
Among the CARS microscopes, a microscope using a broadband light source as Stokes light for spectroscopic detection of generated CARS light is referred to as a multi-color CARS microscope (or multiplex CARS microscope). With the multi-color CARS microscope, it is possible to estimate Raman spectrum from the optical spectrum of the CARS light. In comparison with the method for detecting only a specific spectral component as in the case of Patent Literature 1 (this is referred to as monochromatic CARS or single CARS for the sake of convenience), the amount of acquired information is larger. Accordingly, this microscope is appropriate to more detailed analysis of the measurement object. The basic configuration of the multi-color CARS microscope is shown in 16. (The configuration is based on Patent Literature 2). The output from a short-pulse laser light source 1601 is split into two outputs with a beam splitter 1602. One output is introduced into an optical waveguide such as photonic crystal fiber 1603, and broadband light (referred to as super continuum light) is generated inside. After emission from the fiber, only a desired wavelength component (component having a longer wavelength than that of the pump light) is extracted from the super continuum light with a long-pass filter 1604. The extracted component is used as the Stokes light. The other pump light and the Stokes light are multiplexed with a dichroic mirror 1605 or the like. The multiplexed light is focused on a sample 1606 and irradiated, then CARS light is generated. The generated light is detected with a spectroscope 1607, and the spectrum is acquired.